Bottom Line:
We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data.We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined.In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood.

Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation.

Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Figure 2: Gene content in a region with methylation differences among tissues. Zooming in on a region of chromosome 11 (dashed line) shows tissue-level variation at a locus containing a cluster of genes sharing the leucine-rich repeat (LRR) structural motif. Male and female catkins have a different methylation profile from other tissue types, and an apparent inverse pattern relative to each other over this region.

Mentions:
Fewer MeDIP-seq reads mapped to chromosomal regions where gene density was higher (Additional file 8). Several high-coverage regions also displayed high inter-tissue variability (Figures 1 and 2, Additional file 9). Eleven of the 19 chromosomes (I-IV, VI, VII, X, XI, XV, XVI, XIX) had high coverage by both unique reads and k-mer repeats that indicated possible centromeric or pericentromeric regions (Additional file 8). In all eleven cases, these regions corresponded to putative centromeres identified on the basis of high repeat-to-gene ratios that were also correlated with recombination valleys (P. Ranjan and G. Slavov, pers. comms.). In addition, our k-mer repeat maps correlated well with their equivalent "ambiguous reads" maps, for which no pre-selection process had been done prior to sequencing; this further supports our finding that genome regions with k-mer repeats tend to be more highly methylated than regions with uniquely mapped reads.

Figure 2: Gene content in a region with methylation differences among tissues. Zooming in on a region of chromosome 11 (dashed line) shows tissue-level variation at a locus containing a cluster of genes sharing the leucine-rich repeat (LRR) structural motif. Male and female catkins have a different methylation profile from other tissue types, and an apparent inverse pattern relative to each other over this region.

Mentions:
Fewer MeDIP-seq reads mapped to chromosomal regions where gene density was higher (Additional file 8). Several high-coverage regions also displayed high inter-tissue variability (Figures 1 and 2, Additional file 9). Eleven of the 19 chromosomes (I-IV, VI, VII, X, XI, XV, XVI, XIX) had high coverage by both unique reads and k-mer repeats that indicated possible centromeric or pericentromeric regions (Additional file 8). In all eleven cases, these regions corresponded to putative centromeres identified on the basis of high repeat-to-gene ratios that were also correlated with recombination valleys (P. Ranjan and G. Slavov, pers. comms.). In addition, our k-mer repeat maps correlated well with their equivalent "ambiguous reads" maps, for which no pre-selection process had been done prior to sequencing; this further supports our finding that genome regions with k-mer repeats tend to be more highly methylated than regions with uniquely mapped reads.

Bottom Line:
We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data.We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined.In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood.

Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation.

Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.